NPHS2 gene involved in the steroid-resistant nephrotic syndrome, protein encoded by said gene and diagnostic and therapeutic uses

ABSTRACT

The invention concerns a novel gene, called NPHS2 gene coding for a protein involved in the cortico-resistant nephrotic syndrome, and diagnostic and therapeutic uses of the novel identified nucleotide sequences and amino acids.

[0001] The present invention relates to a novel gene, called NPHS2,which encodes a protein involved in the steroid-resistant nephroticsyndrome.

[0002] Idiopathic nephrotic syndrome is a pathological condition whichappears mainly in children, and which is characterized by massiveproteinurea and nonspecific histological changes in the kidney,sometimes including focal segmental glomerulosclerosis (FSGS). Thesecharacteristics are associated with a diffuse effacing of the pedicelsof podocytes, observed by electron microscopy (Broyer et al., 1998),which reveals nephrotic syndromes whatever their cause. Most casescorrespond to steroid-based therapy and have a good prognosis, butapproximately 20% are resistant to steroids and progress to terminalrenal insufficiency, leading to complete glomerulosclerosis. Referenceis then made to steroid-resistant nephrotic syndrome.

[0003] The ultrafiltration of macromolecules of the plasma duringprimary urine formation in the glomerulus is one of the centralfunctions of the human kidney. The structurally complex capillary wallwhich is responsible for this function is composed of a basal membranecovered with a fenestrated endothelium on its inner surface and withspecialized epithelial cells (podocytes) which form pedicels on theouter surface. In a large number of acquired or hereditary diseases, adysfunctioning of the glomerular filter is observed, resulting inexcessive loss of plasma proteins, leading to a nephrotic syndrome andthen possibly to terminal renal insufficiency.

[0004] The study of genetic diseases which affect the filtration barrierprovides useful models for understanding the physiopathology of theglomerular filtration process. Several of these hereditary disorderswith proteinurea and nephrotic syndrome have been described. The mostsevere is the congenital nephrotic syndrome of the Finnish type (CNF),which is an autosomal recessive disease with strong proteinurea inutero, a nephrotic syndrome at birth usually leading to terminal renalinsufficiency during the first two years of life. CNF is caused bymutations in the NPHS1 gene (Kestila, 1998). Moreover, cases of familialproteinurea or of nephrotic syndrome with histological, focal segmentalglomerulosclerosis (FSGS) lesions have been described in older patients,in particular having reached adult age. Two genetic loci for autosomaldominant FSGS have been mapped, respectively, on the 19q13 locus closeto the locus of the NPHS1 gene (Mathis et al., 1998) and on the11q21-q22 locus (Winn et al., 1999).

[0005] In 1995, a novel steroid-resistant nephrotic syndrome entity forwhich transmission is autosomal recessive was characterized according tothe following criteria: early beginning between three months and fiveyears old, resistance to steroid-based therapy, progression to terminalrenal insufficiency before the age of ten, absence of recurrence afterrenal transplant and absence of any extrarenal disorder. Histologically,only minimal modifications are observed in early biopsies, but FSGS isgenerally present at subsequent stages. A genetic locus involved in thissteroid-resistant nephrotic syndrome has been mapped in the 1q25-q31region between the markers D1S452 and D1S466, this region extending overapproximately 12 cM (Fuchshuber, 1995). This localization has beenconfirmed by another team (Lench et al., 1998) and, more recently, alinkage to this region has also been demonstrated in a family exhibitingan FSGS beginning at adult age (Tsukaguchi et al., 1999).

[0006] The authors of the present invention have now succeeded inprecisely identifying a novel gene involved in the steroid-resistantnephrotic syndrome entity described above. This gene was first calledSRN1 and was then renamed NPHS2.

[0007] A sequence listing is attached, in which the sequence SEQ ID no 1represents the fragment of cDNA of the NPHS2 gene in humanscorresponding to the open reading frame (ORF). This ORF contains 1149bases and encodes a 383 amino acid protein named podocin, the sequenceof which is presented in SEQ ID no 2.

[0008] The sequences SEQ ID no 3 to SEQ ID no 10 represent fragments ofthe genomic DNA of the human NPHS2 gene including, respectively, 8 exons(in bold characters in the attached listing), as follows:

[0009] SEQ ID no 3:

[0010] There are 683 base pairs before the ATG. The cDNA clones obtainedby screening a human fetal kidney cDNA library (Clontech library clonedinto the λgt11 phage) generally begin between bases 615 and 619. Thereare 274 base pairs from the ATG to the splicing site (exon 1), and then147 base pairs of intron sequences.

[0011] SEQ ID no 4:

[0012] There are 151 base pairs of intron, then 104 base pairs of coding(exon 2), and then 123 base pairs of intron.

[0013] SEQ ID no 5:

[0014] There are 336 base pairs of intron, then 73 base pairs of coding(exon 3), and then 291 base pairs of intron.

[0015] SEQ ID no 6:

[0016] There are 187 base pairs of intron, then 83 base pairs of coding(exon 4), and then 90 bp of intron.

[0017] SEQ ID no 7:

[0018] There are 250 base pairs of intron, then 204 base pairs of coding(exon 5), and then 195 base pairs of intron.

[0019] SEQ ID no 8:

[0020] There are 367 base pairs of intron, then 56 base pairs of coding(exon 6), and then 169 base pairs of intron.

[0021] SEQ ID no 9:

[0022] There are 327 bp of intron, then 79 base pairs of coding (exon7), and then 310 base pairs of intron.

[0023] SEQ ID no 10:

[0024] There are 285 base pairs of intron, then 911 base pairs of cDNAsequence up to the polyadenylation site used (exon 8). The stop codon isat position 562, followed by 109 base pairs of additional genomicsequences covering the other potential polyadenylation sites.

[0025] The sequence SEQ ID no 11 covers part of exon 5, exons 6 and 7and a large part of exon 8 (from base 1792 of the cDNA).

[0026] The sequences SEQ ID no 12 to no 27 are primers which are of usefor amplifying human sequences.

[0027] The sequence SEQ ID no 28 is the rat podocin cDNA sequence, thesequence SEQ ID no 29 being the corresponding amino acid sequence.

[0028] A subject of the present invention is therefore an isolatednucleic acid, the sequence of which is chosen from SEQ ID no 3 to SEQ IDno 10, or a homologous sequence defined as

[0029] i) a sequence which is identical to at least 70% of the sequenceSEQ ID no 3 to SEQ ID no 10; or

[0030] ii) a sequence which hybridizes with the sequence SEQ ID no 3 toSEQ ID no 10, or the sequences complementary thereto, under stringenthybridization conditions.

[0031] A subject of the present invention is also an isolated nucleicacid comprising the sequence SEQ ID no 1 or 28, or a homologous sequencedefined as

[0032] i) a sequence which is identical to at least 70% of the sequenceSEQ ID no 1; or

[0033] ii) a sequence which hybridizes with the sequence SEQ ID no 1, orthe sequence complementary thereto, under stringent hybridizationconditions; or

[0034] iii) a sequence which encodes the polypeptide, named podocin, asdefined above.

[0035] Preferably, a homologous nucleotide sequence according to theinvention is identical to at least 75% of the sequences SEQ ID no 1 orSEQ ID no 3 to 10 and 28, more preferably to at least 85%, or to atleast 90%.

[0036] Preferentially, such a homologous nucleotide sequence hybridizesspecifically to the sequences complementary to the sequences SEQ ID nos1, 3 to 10 and 28, under stringent conditions. The parameters whichdefine the conditions of stringency depend on the temperature at which50% of the paired strands separate (Tm).

[0037] For sequences comprising more than 30 bases, Tm is defined by theequation: Tm=81.5+0.41(% G+C)+16.6 Log (concentration of cations)−0.63(%formamide)−(600/number of bases) (Sambrook et al., 1989).

[0038] For sequences less than 30 bases in length, Tm is defined by theequation: Tm=4(G+C)+2(A+T).

[0039] Under suitable stringency conditions, at which aspecificsequences do not hybridize, the hybridization temperature isapproximately 5 to 30° C., preferably 5 to 10° C., below Tm, and thehybridization buffers used are preferably solutions of high ionicstrength, such as a 6×SSC solution for example.

[0040] A nucleotide sequence homologous to the ORF represented in SEQ IDno 1 or 28 includes any nucleotide sequence which differs from thesequence SEQ ID no 1 or 28 by mutation, insertion, deletion orsubstitution of one or more bases, or by the degeneracy of the geneticcode, provided that it encodes a polypeptide which has the biologicalactivity of podocin, as defined below.

[0041] Included among such homologous sequences are the sequences of thegenes, encoding podocin, of mammals other than humans, preferably of aprimate, or of a bovine, a member of the sheep family or a pig, or elseof a rodent, and also the allelic variants or polymorphic sequences.

[0042] The table below gives a certain number of polymorphismsidentified in the NPHS2 gene: Polymorphisms identified in the same NPHS2gene Position on the Exon Polymorphism sequences listed 1 −51/ATG T > G +19 on SEQ ID n° 1 nt 102 (G > A) = G34G  +171 on SEQ ID n° 1 2 nt 288(G > T) = S96S  +357 on SEQ ID n° 1 5 nt 686 (G > A) = R229Q  +755 onSEQ ID n° 1 7 873 +7 A > G  +413 on SEQ ID n° 9 8 nt 954 (T > C) = A318A+1023 on SEQ ID n° 1 nt 1038 (A > G) = L346L +1107 on SEQ ID n° 1

[0043] A subject of the present invention is also an isolatedpolypeptide, named podocin, comprising the amino acid sequence SEQ ID no2 or 29, or a homologous sequence defined as

[0044] i) a sequence which is identical to at least 70% of the sequenceSEQ ID no 2 or 29; or

[0045] ii) a sequence which is encoded by a homologous nucleic acidsequence as defined in claim 2 ii), i.e. a nucleic acid sequence whichhybridizes with is the sequence SEQ ID no 2 or 29, or the sequencecomplementary thereto, under stringent hybridization conditions.

[0046] More generally, the expression “homologous amino acid sequence”is intended to mean any amino acid sequence which differs from thesequence SEQ ID no 2 or 29 by substitution, deletion and/or insertion ofan amino acid or of a small number of amino acids, in particular bysubstitution of natural amino acids with unnatural amino acids orpseudoamino acids, at positions such that these modifications do notsignificantly harm the biological activity of the podocin. Saidsubstitutions are preferably conservative substitutions, i.e.substitutions of amino acids of the same class, such as substitutions ofamino acids with uncharged side chains (such as asparagine, glutamine,serine, threonine and tyrosine), of amino acids with basic side chains(such as lysine, arginine and histidine), of amino acids with acid sidechains (such as aspartic acid and glutamic acid), or amino acids withapolar side chains (such as glycine, alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan andcysteine).

[0047] Preferably, such a homologous amino acid sequence is identical toat least 85% of the sequence SEQ ID no 2 or 29, preferably to at least95%.

[0048] Homology is generally determined using a sequence analysisprogram (for example, Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705). Similar amino acid sequencesare aligned so as to obtain the maximum degree of homology (i.e.identity). For this purpose, it may be necessary to artificiallyintroduce gaps into the sequence. Once the optimal alignment has beenproduced, the degree of homology (i.e. identity) is established byrecording all the positions for which the amino acids of the twocompared sequences are identical, relative to the total number ofpositions.

[0049] The expression “the biological activity of podocin” refers to themaintaining of the integrity of the glomerular filter. An absence or adetrimental modification of podocin causes the leaking of proteins atthe level of the glomerulus and, consequently, the appearance ofproteinurea.

[0050] The polypeptide of the present invention may be synthesized usingall the methods well known to those skilled in the art. The polypeptideof the invention may, for example, be synthesized using syntheticchemistry techniques, such as synthesis of the Merrifield type, which isadvantageous for reasons of purity, of antigenic specificity and ofabsence of undesirable byproducts, and for its ease of production.

[0051] A recombinant podocin may also be produced using a method inwhich a vector containing a nucleic acid comprising the sequence SEQ IDno 1 or no 28, or a homologous sequence, is transferred into a host cellwhich is cultured under conditions which allow the expression of thecorresponding polypeptide.

[0052] The podocin produced may then be recovered and purified.

[0053] The purification methods used are known to those skilled in theart. The recombinant polypeptide obtained may be purified from celllysates and extracts and/or from the culture medium supernatant, viamethods used individually or in combination, such as fractionation,chromatography methods, immunoaffinity techniques using specific mono-or polyclonal antibodies, etc.

[0054] The nucleic acid sequence of interest, encoding podocin, may beinserted into an expression vector, in which it is functionally linkedto elements for regulating the expression thereof, such as in particulartranscription promoters, activators and/or terminators.

[0055] The signals controlling the expression of the nucleotidesequences (promoters, activators, termination sequences, etc.) arechosen as a function of the cellular host used. To this effect, thenucleotide sequences according to the invention may be inserted intovectors which replicate autonomously in the host chosen, or vectorswhich integrate into the host chosen. Such vectors will be preparedaccording to the methods commonly used by those skilled in the art, andthe clones resulting therefrom may be introduced into a suitable hostusing standard methods, such as, for example electroporation or calciumphosphate precipitation.

[0056] The cloning and/or expression vectors as described above,containing one of the nucleotide sequences defined according to theinvention, are also part of the present invention.

[0057] The invention is also directed toward the host cells transfected,transiently or stably, with these expression vectors. These cells may beobtained by introducing, into procaryotic or eucaryotic host cells, anucleotide sequence inserted into a vector as defined above, and thenculturing said cells under conditions which allow replication and/orexpression of the nucleotide sequence transfected.

[0058] Examples of host cells include, in particular, mammalian cells,such as COS-7, 293 or MDCK cells, insect cells, such as SF9 cells,bacteria, such as E. coli, and yeast strains, such as YRG2.

[0059] The various nucleotide sequences of the invention may or may notbe of artificial origin. They may be DNA or RNA sequences, obtained byscreening sequence libraries using probes developed on the basis of thesequences SEQ ID no 1 or 28 and 3 to 10. Such libraries may be preparedusing conventional molecular biology techniques known to those skilledin the art.

[0060] The nucleotide sequences according to the invention may also beprepared by chemical synthesis, or by mixed methods including chemicalor enzymatic modification of sequences obtained by screening libraries.

[0061] These nucleotide sequences make it possible to prepare probes orprimers which hybridize specifically with a sequence SEQ ID no 1 or 28,or 3 to 10, according to the invention, or the strand complementarythereto. Suitable hybridization conditions correspond to the conditionsof temperature and of ionic strength usually used by those skilled inthe art, preferably under stringent conditions as defined above. Theseprobes may be used as an in vitro diagnostic tool, for detecting, viahybridization experiments, in particular “in situ” hybridizationexperiments, transcripts specific for the polypeptide of the inventionin biological samples, or for demonstrating aberrant syntheses orgenetic abnormalities resulting from a polymorphism, from mutations orfrom incorrect splicing.

[0062] The nucleic acids of the invention which are of use as probescomprise a minimum of 10 nucleotides, preferentially at least 20nucleotides, more preferentially at least 100 nucleotides. The nucleicacids which are of use as primers comprise a minimum of 10 nucleotides,preferably at least 14 nucleotides, and preferentially less than 40nucleotides.

[0063] More precisely, a subject of the present invention is a nucleicacid having at least 10 nucleotides, which hybridizes specifically withone of the nucleic acid sequences SEQ ID no 1 or 28, or 3 to 10, or thesequence complementary thereto, under stringent hybridizationconditions.

[0064] Advantageously, use may be made, as a probe, of the nucleic acidconsisting of the sequence SEQ ID no 11, which covers part of exon 5,exons 6 and 7 and a large part of exon 8 (from base 728 to base 1792 ofthe cDNA).

[0065] Moreover, the nucleic acids consisting of the sequences SEQ ID no12 to SEQ ID no 27 may be used as a primer for an amplification (forexample by PCR).

[0066] Preferentially, the probes or primers of the invention arelabeled prior to their use. For this, several techniques are within thescope of those skilled in the art, such as, for example, fluorescent,radioactive, chemiluminescent or enzymatic labeling.

[0067] The in vitro diagnostic methods in which these oligonucleotidesare used for detecting mutations or genomic rearrangements, in the NPHS2gene, are included in the present invention.

[0068] Those skilled in the art are well aware of the standard methodsfor analyzing the DNA contained in a biological sample and fordiagnosing a genetic disorder. Many strategies for genotypic analysisare available (Antonarakis et al., 1989; Cooper et al., 1991).

[0069] Preferably, use may be made of the DGGE (denaturing gradient gelelectrophoresis) method, the SSCP (single strand confirmationpolymorphism) method or the DHPLC (denaturing high performance liquidchromatography; Kuklin et al., 1997; Huber et al., 1995) method, fordetecting an abnormality in the NPHS2 gene. Such methods are preferablyfollowed by direct sequencing. The RT-PCR method may advantageously beused to detect abnormalities in the NPHS2 transcript, since it makes itpossible to visualize the consequences of a splicing mutation whichcauses the loss of one or more exons in the transcript, or an aberrantsplicing due to the activation of a cryptic site. This method is alsopreferably followed by direct sequencing. The most recently developedmethods using DNA chips may also be used to detect an abnormality in theNPHS2 gene (Bellis et al., 1997).

[0070] The cloning of the NPHS2 gene and also the identification ofvarious mutations responsible for the steroid-resistant nephroticsyndrome make it possible to envision direct diagnoses. The specificityand reliability of such methods for diagnosis are particularlyappreciable for prenatal diagnosis. The nucleic acid sequences of thepresent invention therefore represent a particularly advantageous toolfor genetic counsel.

[0071] A subject of the present invention is therefore the use of atleast one nucleic acid as defined above, for detecting an abnormality inthe NPHS2 gene, defined as comprising a nucleic acid sequence SEQ ID no3 to 10, or in its transcript, defined as comprising a nucleic acidsequence complementary to the sequence SEQ ID no 1.

[0072] A subject of the invention is, consequently, a method for the invitro diagnosis of a steroid-resistant nephrotic syndrome related to amutation of the NPHS2 gene, comprising the steps consisting in:

[0073] a1) placing a biological sample containing DNA together withspecific oligonucleotides for amplifying all or part of the NPHS2 gene,defined as comprising a nucleic acid sequence chosen from SEQ ID no 3 to10, or a homologous sequence;

[0074] b1) amplifying said DNA;

[0075] c1) detecting the amplification products;

[0076] d1) comparing the amplification products obtained with thoseobtained using a control sample, and detecting in this way a possibleabnormality in said NPHS2 gene, indicating a steroid-resistant nephroticsyndrome related to a mutation of the NPHS2 gene; or, according to analternative,

[0077] a2) placing a biological sample containing RNA together withspecific oligonucleotides for amplifying all or part of the transcriptof the NPHS2 gene, defined as comprising a nucleic acid sequencecomplementary to the sequence SEQ ID no 1, or a homologous sequence;

[0078] b2) amplifying said DNA;

[0079] c2) detecting the amplification products;

[0080] d2) comparing the amplification products obtained with thoseobtained using a control sample, and detecting in this way a possibleabnormality in said transcript of the NPHS2 gene, indicating asteroid-resistant nephrotic syndrome related to a mutation of the NPHS2gene.

[0081] The isolated nucleic acids comprising a sequence which differsfrom the sequence SEQ ID no 1 by a mutation, insertion or deletion, inparticular in at least one of the positions of nucleotides 481, 173/174,488, 924/925, 128, 343, 482, 548, 607 and 940, or else 1033, 529, 622,774-782, 154, 422, 442, 571, 572, 583, 783, 794 and 848, are also partof the invention.

[0082] Among the mutations already identified, the following, given intables 1 and 2, are in particular noted. Table 1 Mutations in the NPHS2gene Effect on the Identification n° Type of coding of the familyMutation Position on mutation^(a) Nucleotide change sequence Exonconcerned status^(b) SEQ ID n° 1 Nonsense C → T at 412 R138X 3 8 H 481Deletion/ Insertion of G at 104/5 Frameshift 1 14 H 173/174 insertionDeletion of G at 419 Frameshift 3 14 H^(c) 488 Deletion of AA at 855/6Frameshift 7 9 h 924/925 Missense C → T at 59 P20L 1 15 H 128 G → T at274 G92C 1 3 h^(c,d) 343 G → A at 413 R138Q 3 4 h^(c) 482 6 H 7 H 11 H12 h 13 H A → C at 479 D160G 4 16 H 548 G → A at 538 V180M 5 10 H 607 12h 940 C → T at 871 R291W 7 2 h^(c)

[0083] TABLE 2 Other mutations in the NPHS2 gene Type of Effect on theNumber of Mutation Position on mutation Nucleotide change^(a) codingsequence Exon families status^(b) SEQ ID n° 1 Nonsense C → T at 964R322X 8 1 h 1033  Deletion/ Insertion T at 460 Frameshift 4 1 h 529insertion Deletion T 553 Frameshift 5 1 h^(c) 622 Deletion 9bp at705-713 Deletion TER236-238 5 1 H 774-782 Missense G → A at 85 A29T 1 11h 154 C → T at 353 P118L 2 1 h 422 G → A at 373 A125I 2 1 h 442 C → A at502 R168S 4 1 h 571 C → T at 502 R168C 4 1 h 571 G → A at 503 R168H 4 1h 572 C → G at 514 L172V 4 1 h 583 G → T at 714 R238S 5 1 h 783 C → T at725 A242V 5 2 h 794 T → A at 779 V260E 6 2 H 848

[0084] These tests can in particular be exploited in families whichalready have an affected child, for presymptomatic diagnosis (inparticular prenatal diagnosis).

[0085] In sporadic cases, the detection of a mutation of the NPHS2 genemakes it possible to modify the treatment (and in particular to avoidimmunosuppressive treatments which will be ineffective) and to predict alack of recurrence after renal transplant.

[0086] This diagnostic test may also be used to investigate theassociation of certain polymorphic variants of podocin in otherpathological conditions with secondary involvement of abnormalities ofthe glomerular filter (diabetic nephropathy, nephropathy in AIDS,nephron loss, arterial hypertension). These variants may representfactors of susceptibility to the triggering or to the progression of thenephropathy in these diseases.

[0087] A subject of the invention is also antibodies directed againstthe podocin polypeptide as defined above.

[0088] They may be poly- or monoclonal antibodies, or fragments thereof,or chimeric antibodies, in particular humanized or immunoconjugatedantibodies.

[0089] The polyclonal antibodies may be obtained from the serum of ananimal immunized against a polypeptide according to the usualprocedures.

[0090] According to one embodiment of the invention, a suitable peptidefragment, which may be coupled via a reactive residue to a protein or toanother peptide, may be used as an antigen. Rabbits are immunized withthe equivalent of 1 mg of the peptide antigen according to the proceduredescribed by Benoit et al. (1982). At four-week intervals, the animalsare given injections of 200 μg of antigen and bled 10 to 14 days later.After the third injection, the antiserum is examined in order todetermine its ability to bind to the antigenic peptide radiolabeled withiodine, which is prepared by the chloramine-T method, and is thenpurified by chromatography on a carboxymethylcellulose (CMC) ionexchange column. The antibody molecules are then recovered from themammals and isolated to the desired concentration by methods well knownto those skilled in the art, for example using DEAE Sephadex to obtainthe IgG fraction.

[0091] In order to increase the specificity of the polyclonal serum, theantibodies may be purified by immunoaffinity chromatography usingsolid-phase immunizing polypeptides. The antibody is brought intocontact with the solid-phase immunizing polypeptide for a sufficientperiod of time so as to cause the polypeptide to immunoreact with theantibody molecule in order to form a solid-phase immunocomplex.

[0092] By way of example, polyclonal antibodies in rabbits were producedagainst two recombinant proteins comprising the fragments of amino acids15 to 89 and 135 to 383 of podocin, coupled to six histidine residues onthe N-terminal side, the cDNAs having been subcloned into the vector PQE32 (Quiagen) and expressed in E. coli.

[0093] Monoclonal antibodies may be obtained according to theconventional method for culturing hybridomas described by Kohler andMilstein (1975).

[0094] The antibodies or antibody fragments of the invention may, forexample, be chimeric antibodies, humanized antibodies, Fab fragments andF(ab′)2 fragments. They may also be in the form of labeled antibodies orimmunoconjugates.

[0095] The antibodies of the invention, in particular the monoclonalantibodies, may especially be used for the immunohistochemical analysisof podocin on specific tissue sections, for example byimmunofluorescence, gold labeling, immunoperoxidase, etc.

[0096] The antibodies thus produced may advantageously be used in anysituation in which the expression of podocin must be observed.

[0097] A subject of the invention is also the use of at least oneantibody thus produced, for detecting or purifying a polypeptide asdefined above in a biological sample.

[0098] More precisely, the invention relates to an in vitro method fordetecting or measuring the level of expression of podocin in abiological sample, comprising bringing at least one antibody as definedabove into contact with said biological sample, under conditions whichallow the possible formation of specific immunocomplexes between thepodocin and said antibody or antibodies, and detecting the specificimmunocomplexes possibly formed. The setting up of such a test (of theELISA type for example) may in particular be of use in searching for thedevelopment of anti-podocin antibodies after renal transplant, incertain autoimmune renal diseases, or even in steroid-sensitivenephrotic syndrome.

[0099] A subject of the invention is also a kit for carrying out thismethod, comprising:

[0100] at least one podocin-specific antibody, optionally attached to asupport;

[0101] means for revealing the formation or specific antigen/antibodycomplexes between the podocin and said antibody, and/or means forquantifying these complexes.

[0102] A subject of the invention is also a pharmaceutical compositioncomprising a podocin polypeptide as defined above or a nucleic acidencoding said polypeptide, in combination with a pharmaceuticallyacceptable vehicle.

[0103] The methods of administration, the dosages and the pharmaceuticalforms of the pharmaceutical compositions according to the invention,containing at least one polypeptide, may be determined in the usual wayby those skilled in the art, in particular according to the criteriagenerally taken into account in establishing a therapeutic treatmentsuitable for a patient, such as, for example, the age or body weight ofthe patient, the seriousness of his or her general condition, thetolerance to the treatment, and the side effects noted, etc.

[0104] In general, a therapeutically or prophylactically effectiveamount ranging from approximately 0.1 μg to approximately 1 mg may beadministered to human adults.

[0105] A subject of the invention is also a pharmaceutical compositioncomprising a nucleic acid as defined above, encoding a polypeptide withpodocin activity, and a pharmaceutically acceptable vehicle, saidcomposition being intended to be used in gene therapy. The nucleic acid,preferably inserted into a generally viral vector (such as adenovirusesand retroviruses), may be administered in naked form, free of anyvehicle promoting transfer to the target cell, such as anionicliposomes, cationic lipids, microparticles, for example goldmicroparticles, precipitating agents, for example calcium phosphate, orany other agent facilitating transfection. In this case, thepolynucleotide may simply be diluted in a physiologically acceptablesolution, such as a sterile solution or a sterile buffer solution, inthe presence or absence of a vehicle.

[0106] Alternatively, a nucleic acid of the invention may be associatedwith agents which facilitate transfection. It may, inter alia, be (i)associated with a chemical agent which modifies cellular permeability,such as bupivacaine; (ii) encapsulated in liposomes, optionally in thepresence of additional substances which facilitate transfection; or(iii) associated with cationic lipids or microparticles made of silica,of gold or of tungsten.

[0107] When the nucleic acid constructs of the invention covermicroparticles, these microparticles may be injected intradermally orintraepidermally using the gene gun technique (WO 94/24263).

[0108] The amount to be used as a medicinal product depends inparticular on the nucleic acid construct itself, on the individual towhich this nucleic acid is administered, on the method of administrationand the type of formulation, and on the pathological condition. Ingeneral, a therapeutically or prophylactically effective amount rangingfrom approximately 0.1 μg to approximately 1 mg, preferably fromapproximately 1 μg to approximately 800 μg, and preferentially fromapproximately 25 μg to approximately 250 μg, can be administered tohuman adults.

[0109] The nucleic acid constructs of the invention may be administeredvia any conventional route of administration, such as in particularparenterally. The choice of the route of administration depends inparticular on the formulation chosen. An administration targeted to therenal tissue, in particular to the glomeruli, may be particularlyadvantageous.

[0110] The polypeptide of the invention, or the nucleic acid encodingthis polypeptide, is of use as a medicinal product, especially for thetreatment of a renal disease, in particular for the treatment of asteroid-resistant nephrotic syndrome related to a mutation of the NPHS2gene or occurring in the context of a general disease (AIDS, diabetes,etc.).

[0111] Finally, a subject of the invention is therefore a method oftherapeutic treatment, in which an effective amount of a podocinpolypeptide as defined above or a nucleic acid encoding this polypeptideis administered to a patient requiring such a treatment.

[0112] The patient targeted is generally a human, but the applicationmay also be extended to any mammal, where appropriate.

[0113] The following examples and also the attached figure illustratethe invention without limiting the scope thereof.

Legend to the Figure

[0114] The attached figure is a map of the NPHS2 region. The 2.5 Mbcandidate region is delimited by the markers D1S1640 and 183f10-CA. Theposition on the map of the polymorphic markers, of the STS sequences (inbold characters and italics), of the unique EST sequences and of theUniGene EST clusters (in normal characters) is indicated. The YACs, PACsand cosmids are represented by lines. The genes are indicated by hatchedboxes. NGAP is represented by two boxes separated by a horizontal linewhich symbolizes the presence of the exons which are alternativelyspliced in the 5′ position probably due to an alternative promoter.RPS14P, a pseudogene of the ribosomal protein S14, is located inside theNGAP intron.

EXAMPLES Example 1

[0115] Identification of the NPHS2 Gene

[0116] The approach used by the authors of the present invention inorder to identify the NPHS2 gene was to define the minimum geneticinterval in which the gene is located, then to establish the physicalmap of the region by constructing a PAC contig covering the region, tocarry out an inventory of the known genes and of the ESTs of the regionand to characterize the ESTs (by RACE-PCR and screening a fetal kidneycDNA library).

[0117] 1 Physical Mapping of the Candidate Region and Localization ofthe NPHS2 Gene:

[0118] A linkage analysis using microsatellite markers (Dib et al.,1996) and also new families of patients made it possible to localize theNPHS2 locus between the markers D1S480 and D1S2883. A YAC contig (20clones) covering the region between these two markers was constructed. AP1 artificial chromosome (PAC) contig was also constructed so as tocover this region estimated at approximately 3 Mb. It was then possibleto characterize other microsatellite markers in this contig. Twofamilies exhibiting the combination events made it possible to preciselylocalize the locus for the disease between D1S1640 and 183F10CA, a newmicrosatellite marker identified by sequencing subclones of the region.The 35 PAC contig between these two markers covers approximately 2 to2.5 Mb, but contains 5 gaps partially filled with 14 cosmids.

[0119] The authors of the invention then located on this contig, bysearching in the databanks for sequences potentially localized in theregion and sequencing the ends of the YACs, of the PACs and of thecosmids, and also of the subclones of various PACs potentiallycontaining CpG islands, genes already known, (UniGene) EST clusters andindependent ESTs.

[0120] 2. Identification of the NPHS2 Gene

[0121] In consulting the Sanger Centre database, it was found that thePAC 545A16 contained the marker D1S215 localized close to the telomericedge of the region of interest, as did the EST AA398634, which came froma testes library and contained short sequences weakly homologous to thestomatin gene, but curiously, a priori, in the direction opposite to theEST. The authors of the invention then localized this EST on the cosmid28e17 and on the PAC 302d13 and showed, by RT-PCR, that it was expressedin the kidney.

[0122] Multiple attempts of RACE-PCR were then necessary in order toobtain a cDNA from this EST. In fact, the products obtainedcorresponded, in most of the experiments, to genomic DNAs which appearedto be unspliced. However, one of the products obtained corresponded to atranscript containing a short open reading frame and homologous to sixESTs (Unigene cluster Hs. 192657) all originating from a human kidneylibrary, but which had not been localized on the genome. In fact, it sohappens that the ESTs of the UniGene cluster Hs. 254975, to which theEST AA398634 belongs, appear to belong to another gene, or pseudogene,the direction of transcription of which is opposite to NPHS2, and whichpartially overlaps the 3′ sequence of NPHS2, which explains the dataprovided by the databanks relating to the EST AA398634. Using thisRACE-PCR product described above as a probe to hybridize a Northern blotcontaining RNAs from various tissues, it was shown that this transcriptof approximately 2 kb was expressed only in the kidney. These resultswere confirmed by hybridizing a dot blot containing RNAs from 50different tissues (Clontech). A strong signal was obtained only withadult kidney and fetal kidney. The localization of this gene on thecontig and its virtually exclusive expression in the kidney made thisgene an excellent candidate gene, this hypothesis being reinforced bythe virtual absence of a product of amplification by RT-PCR, usingprimers located both in the 5′ part and in the 3′ part of the cDNA, withthe terminal kidney-extracted RNA of a patient. The complete cDNA of theNPHS2 gene was cloned by screening a human fetal kidney cDNA librarywith the probe used to hybridize the Northern Blot (sequence ID no 11).

[0123] The intron-exon junctions and the genomic sequences upstream ofexon 1 were obtained by direct sequencing of the PAC 302d13 and of thecosmid 28e17.

Example 2

[0124] Identification of Mutations in Families of Patients

[0125] Having characterized the intron-exon structure of the gene, theauthors of the invention then sought, by SSCP (Single StrandConformation Polymorphism), mutations in 16 unrelated patientsexhibiting a familial steroid-resistant nephrotic syndrome as describedabove (early beginning, rapid progression to terminal renalinsufficiency, no recurrence after transplant and focal segmentalglomerulosclerosis on renal biopsies) and belonging to families in whichthe study of the haplotypes was compatible with a linkage to the NPHS2locus.

[0126] For this SSCP analysis, the exons were amplified by PCR usingflanking intron primers. The PCR conditions and the primers were chosenusing the program Oligo 5.0 (NBI), and were as follows:

[0127] exon 1, 5′-GCA GCG ACT CCA CAG GGA CT-3′ (SEQ ID no 12) and5′-TCA GTG GGT CTC GTG GGG AT-3′ (SEQ ID no 13);

[0128] exon 2, 5′-AGG CAG TGA ATA CAG TGA AG-3′ (SEQ ID no 14) and5′-GGC CTC AGG AAA TTA CCT A-3′ (SEQ ID no 15);

[0129] exon 3, 5′-TTC TGG GAG TGA TTT GAA AG-3′ (SEQ ID no 16) and5′-TGA AGA AAT TGG CAA GTC AG-3′ (SEQ ID no 17);

[0130] exon 4, 5′-AAG GTG AAA CCC AAA CAG C-3′ (SEQ ID no 18) and 5′-CGGTAG GTA GAC CAT GGA AA-3′ (SEQ ID no 19);

[0131] exon 5, 5′-CAT AGG AAA GGA GCC CAA GA-3′ (SEQ ID no 20) and5′-TTT CAG CAT ATT GGC CAT TA-3′ (SEQ ID no 21);

[0132] exon 6, 5′-CTC CCA CTG ACA TCT GA-3′ (SEQ ID no 22) and 5′-AATTTA AAA TGA AAC CAG AA-3′ (SEQ ID no 23);

[0133] exon 7, 5′-CTA AAT CAT GGC TGC ACA CC-3′ (SEQ ID no 24) and5′-CTT CCT AAA GGG CAG TCT GG-3′ (SEQ ID no 25);

[0134] exon 8, 5′-GGT GAA GCC TTC AGG GAA TG-3′ (SEQ ID no 26) and5′-TTC TAT GGC AGG CCC CTT TA-3′ (SEQ ID no 27);

[0135] at hybridization temperatures of 50° C. (exon 6), 55° C. (exons2, 3, 4 and 5) and 60° C. (exons 1, 7 and 8). Because of the high GCcontent of exon 1, the PCR was carried out using Qiagen Taq polymeraseand Q-solution according to the manufacturer's instructions. Inaddition, because of its size, the exon 1 PCR product had to be digestedinto two fragments with the SmaI enzyme, before the gel electrophoresis.The migration was performed for two hours at 600 V, 25 mA and 15 W withthe Genephor Electrophoresis Unit, using the GeneGel Excel 12.5/24 kit(Pharmacia). The staining was carried out with a “GeneStain AutomatedGel Stainer” using the PlusOne Silver Staining kit (Pharmacia).

[0136] Results

[0137] Ten different mutations were observed. Some result in aframeshift or in the appearance of a premature stop codon and aretherefore inactivating mutations, which proves that the gene identifiedis indeed the NPHS2 gene. Others are missense mutations occurring invery conserved regions of the protein, segregating in the families withthe disease, and not found in 80 control chromosomes, which stronglysuggests that these mutations are indeed responsible for the phenotypein the affected children.

[0138] One of the missense mutations (R138Q) was found in sixindividuals who were not related but who came from the same part ofEurope, suggesting the possibility of a founder effect for thismutation.

Example 3

[0139] Study of Expression of the NPHS2 Gene in the kidney by in situHybridization

[0140] Method

[0141] The paraffin was removed from paraffin-covered 6-μm kidneysections, which were rehydrated and then microwave-treated in sodiumcitrate buffer (0.01 M, pH 6) in order to increase the hybridizationsignal. The NPHS2 riboprobes were synthesized from the PCR product of1065 base pairs (position 728 to 1792 of the NPHS2 cDNA, SEQ ID no 1)subcloned into the vector PGEM-Teasy. The antisense probe wassynthesized, after digestion with SalI, using T7 polymerase and thesense probe was synthesized, after digestion with SacII, using Sp6polymerase. The riboprobes were labeled either with digoxigenin-11-UTP(Boehringer Mannheim) according to the manufacturer's instructions, orwith [³⁵S]UTP as described in Sibony et al., 1995. In situ hybridizationexperiments were carried out as described in Kalatzis et al. (1998) andHeidet et al. (1997) for digoxigenin-11-UTP and [³⁵S]UTP probes,respectively.

[0142] Results

[0143] These in situ hybridization experiments made it possible to showthat the NPHS2 gene was expressed only in the podocytes in the maturekidney. In fetal kidneys, no signal was observed at the early stages ofdevelopment of the nephron. On the other hand, strong signals weredetected in the lower segment of the S-shaped body, in the regioncorresponding to the future podocytes. This expression persists in theimmature glomeruli and in the mature glomeruli of the , deep cortex.These results, which show the exclusive expression of the NPHS2 gene inthe podocytes, both early during development and in the matureglomeruli, are entirely in agreement with the pathology observed andjustify the name “podocin” for the protein encoded by the NPHS2 gene.

Bibliography

[0144] Antonarakis S. E., Diagnosis of genetic disorders at the DNAlevel. N Engl. J. Med. 320:153-163 (1989).

[0145] Antonarakis, S. E., Recommendations for a nomenclature system forhuman gene mutations. Nomenclature Working Group. Hum. Mut. 11, 1-3(1998).

[0146] Bellis et al., medecine/sciences, 13:1317-24, (1997).

[0147] Benoit et al., PNAS USA, 79, 917-921 (1982).

[0148] Broyer M., Meyrier A., Niaudet P. & Habib R. Minimal changes andfocal segmental glomerular sclerosis. In Oxford Textbook of ClinicalNephrology 2^(nd) ed. (eds Davison A. M. et al.) 493-535 (OxfordUniversity Press, Inc., 1998).

[0149] Cooper et al., Diagnosis of genetic disease using recombinantDNA, 3^(rd) Edition, Hum Genet., 87:519-560 (1991).

[0150] Conton, P. J. et al., Clinical and pathologic features offamilial focal segmental glomerulosclerosis. Am. J. Kidney Dis. 26,34-40 (1995).

[0151] Dib, C. et al. A comprehensive genetic map of the human genomebased on 5,264 microsatellites. Nature 380, 152-154 (1996).

[0152] Fuchshuber, A. et al. Mapping a gene (NPHS2) to chromosome1q25-q31 in idiopathic nephrotic syndrome confirms a distinct entity ofautosomal recessive nephrosis. Hum. Mol. Genet. 4, 2155-2158 (1995).

[0153] Heidet, L. et al. Diffuse leiomyomatosis associated with X-linkedAlport syndrome: extracellular matrix study using immunohistochemistryand in situ hybridization. Lab. Invest. 76, 233-243 (1997).

[0154] Huber, C. G. et al., Rapid and accurate sizing of DNA fragmentsby ion-pair chromatography on alkylated nonporouspoly(styrenedivinylbenzene) particles. Anal. Chem. 67, 578-585 (1995).

[0155] Kuklin, A. et al., Detection of single-nucleotide polymorphismswith the WAVE™ DNA fragment analysis system. Genetic Testing 1, 201-206(1997/98).

[0156] Kalatzis, V., Sahly, I., El-Amraoui, A. & Petit, C. Eyalexpression in the developing ear and kidney: towards the understandingof the pathogenesis of Branchio-Oto-Renal (BOR) syndrome. Dev. Dyn. 213,486-499 (1998).

[0157] Kestilä, M. et al. Positionally cloned gene for a novelglomerular protein-nephrin-is mutated in congenital nephrotic syndrome.Mol. Cell 1, 575-582 (1998)

[0158] Köhler and Milstein, Nature, 256, 495-497, (1975).

[0159] Lench et al., Am. J. Hum. Genet. 63, A296 (1998).

[0160] Mathis, B. J. et al. A locus for inherited focal segmentalglomerulosclerosis maps to chromosome 19q13. Kidney. Int. 53, 282-286(1998).

[0161] Sambrook et al., Molecular cloning, a laboratory manual SpringHarbor Laboratory Press, 9.54-62 (1989)

[0162] Tsukaguchi et al., Adult onset familial FSGS mapping tochromosome 1q. J. Am. Soc. Nephrol. 10, 443A (1999).

[0163] Winn, M. P. et al. Linkage of a gene causing familial focalsegmental glomerulosclerosis to chromosome 11 and further evidence ofgenetic heterogeneity. Genomics 58, 113-120 (1999).

1 29 1 1853 DNA Homo sapiens CDS (70)..(1221) 1 cacagggact gcgctcccttgcccctagcg ctcccgcgct gctgctccag ccgcccggca 60 gctctgagg atg gag agg agggcg cgg agc tcc tcc agg gag tcc cgc ggg 111 Met Glu Arg Arg Ala Arg SerSer Ser Arg Glu Ser Arg Gly 1 5 10 cga ggc ggc agg act ccg cac aag gagaac aag agg gca aag gcc gag 159 Arg Gly Gly Arg Thr Pro His Lys Glu AsnLys Arg Ala Lys Ala Glu 15 20 25 30 agg agc ggc ggg ggc cgc ggg cgc caggag gct ggg ccc gag ccg tcg 207 Arg Ser Gly Gly Gly Arg Gly Arg Gln GluAla Gly Pro Glu Pro Ser 35 40 45 ggc tcc gga cgg gcg ggg acc ccg ggg gagccc cga gcg ccc gcc gcc 255 Gly Ser Gly Arg Ala Gly Thr Pro Gly Glu ProArg Ala Pro Ala Ala 50 55 60 acg gtg gtg gac gtg gat gag gtc cga ggc tccggc gag gag ggc acc 303 Thr Val Val Asp Val Asp Glu Val Arg Gly Ser GlyGlu Glu Gly Thr 65 70 75 gag gtg gtg gcg ctg ttg gag agc gag cgg ccc gaggaa ggt acc aaa 351 Glu Val Val Ala Leu Leu Glu Ser Glu Arg Pro Glu GluGly Thr Lys 80 85 90 tcc tcc ggc tta ggg gcc tgt gag tgg ctt ctt gtc ctcatt tcc ctg 399 Ser Ser Gly Leu Gly Ala Cys Glu Trp Leu Leu Val Leu IleSer Leu 95 100 105 110 ctc ttc atc atc atg acc ttc cct ttt tcc atc tggttc tgc gta aag 447 Leu Phe Ile Ile Met Thr Phe Pro Phe Ser Ile Trp PheCys Val Lys 115 120 125 gtt gta caa gag tat gaa aga gta att ata ttc cgactg gga cat ctg 495 Val Val Gln Glu Tyr Glu Arg Val Ile Ile Phe Arg LeuGly His Leu 130 135 140 ctt cct gga aga gcc aaa ggc cct ggt ctt ttc tttttt ttg ccc tgc 543 Leu Pro Gly Arg Ala Lys Gly Pro Gly Leu Phe Phe PheLeu Pro Cys 145 150 155 ctg gat acc tac cac aag gtt gac ctt cgt ctc caaact ctg gag ata 591 Leu Asp Thr Tyr His Lys Val Asp Leu Arg Leu Gln ThrLeu Glu Ile 160 165 170 cct ttt cat gag atc gtg acc aaa gac atg ttt ataatg gag ata gat 639 Pro Phe His Glu Ile Val Thr Lys Asp Met Phe Ile MetGlu Ile Asp 175 180 185 190 gcc att tgc tac tac cga atg gaa aat gcc tctctt ctc cta agc agt 687 Ala Ile Cys Tyr Tyr Arg Met Glu Asn Ala Ser LeuLeu Leu Ser Ser 195 200 205 ctt gct cat gta tct aaa gct gtg caa ttc cttgtg caa acc act atg 735 Leu Ala His Val Ser Lys Ala Val Gln Phe Leu ValGln Thr Thr Met 210 215 220 aag cgt ctc cta gca cat cga tcc ctc act gaaatt ctt cta gag agg 783 Lys Arg Leu Leu Ala His Arg Ser Leu Thr Glu IleLeu Leu Glu Arg 225 230 235 aag agc atc gcc caa gat gca aag gtt gcc ttggat tca gtg acc tgt 831 Lys Ser Ile Ala Gln Asp Ala Lys Val Ala Leu AspSer Val Thr Cys 240 245 250 att tgg gga atc aaa gtg gag aga ata gaa attaaa gat gtg agg ttg 879 Ile Trp Gly Ile Lys Val Glu Arg Ile Glu Ile LysAsp Val Arg Leu 255 260 265 270 cca gct ggg ctt cag cac tca ctg gct gtggag gct gaa gcg caa aga 927 Pro Ala Gly Leu Gln His Ser Leu Ala Val GluAla Glu Ala Gln Arg 275 280 285 caa gcc aaa gtg cgg atg att gct gca gaagcg gaa aag gct gct tct 975 Gln Ala Lys Val Arg Met Ile Ala Ala Glu AlaGlu Lys Ala Ala Ser 290 295 300 gag tcc ctg agg atg gca gct gag att ctgtca ggc acc cct gct gct 1023 Glu Ser Leu Arg Met Ala Ala Glu Ile Leu SerGly Thr Pro Ala Ala 305 310 315 gtt cag ctt cga tac ctc cac acc ctt cagtct ctg tcc aca gag aag 1071 Val Gln Leu Arg Tyr Leu His Thr Leu Gln SerLeu Ser Thr Glu Lys 320 325 330 cct tcc act gtg gtt tta cct ttg cca tttgac cta ctg aat tgc ctg 1119 Pro Ser Thr Val Val Leu Pro Leu Pro Phe AspLeu Leu Asn Cys Leu 335 340 345 350 tct tct ccc agc aac aga act cag ggaagc ctc ccc ttc cca agt cct 1167 Ser Ser Pro Ser Asn Arg Thr Gln Gly SerLeu Pro Phe Pro Ser Pro 355 360 365 tcc aaa cct gtt gag cca cta aat cctaaa aag aaa gac tct ccc atg 1215 Ser Lys Pro Val Glu Pro Leu Asn Pro LysLys Lys Asp Ser Pro Met 370 375 380 tta tag gaaggatggg gcataatgtgactgtaaagg ggcctgccat agaaaagtca 1271 Leu catccctgag ggagacactctgtcctcatt ccctgccctt cctttggttg ccatatggaa 1331 tggccatgga atgcacgaagtcacaatgca ccatccatga gaagacrgtg aaatgatgta 1391 atgacagaga aggcagacaacatgtttccg tgactcatct agtcagagca attatgggaa 1451 acagctttgg tcaacattctactttggaaa gaattttgag tctagatgtg gttaaatttt 1511 gacttctggg aacttggttcagatgtccct ttcactgtat gtcctctgac ccctttggca 1571 aggttgccac agctcccacagcccttccta caagcaccta tcattgggct tgtcacactc 1631 tattgctctt ctgtcccgaagatgcagtct tctctccaat gatactacca agtcttagtt 1691 ttcctcaacc acactcaatctttttgctcc accctgaatt cctcacacct aaccctgata 1751 gttacctaaa gtgacacttaaatgtttcag agtgaatgca aaaaagagag atgtacttgg 1811 agtcggatat acaatttatccctaattaaa gcatttaaaa gg 1853 2 383 PRT Homo sapiens 2 Met Glu Arg ArgAla Arg Ser Ser Ser Arg Glu Ser Arg Gly Arg Gly 1 5 10 15 Gly Arg ThrPro His Lys Glu Asn Lys Arg Ala Lys Ala Glu Arg Ser 20 25 30 Gly Gly GlyArg Gly Arg Gln Glu Ala Gly Pro Glu Pro Ser Gly Ser 35 40 45 Gly Arg AlaGly Thr Pro Gly Glu Pro Arg Ala Pro Ala Ala Thr Val 50 55 60 Val Asp ValAsp Glu Val Arg Gly Ser Gly Glu Glu Gly Thr Glu Val 65 70 75 80 Val AlaLeu Leu Glu Ser Glu Arg Pro Glu Glu Gly Thr Lys Ser Ser 85 90 95 Gly LeuGly Ala Cys Glu Trp Leu Leu Val Leu Ile Ser Leu Leu Phe 100 105 110 IleIle Met Thr Phe Pro Phe Ser Ile Trp Phe Cys Val Lys Val Val 115 120 125Gln Glu Tyr Glu Arg Val Ile Ile Phe Arg Leu Gly His Leu Leu Pro 130 135140 Gly Arg Ala Lys Gly Pro Gly Leu Phe Phe Phe Leu Pro Cys Leu Asp 145150 155 160 Thr Tyr His Lys Val Asp Leu Arg Leu Gln Thr Leu Glu Ile ProPhe 165 170 175 His Glu Ile Val Thr Lys Asp Met Phe Ile Met Glu Ile AspAla Ile 180 185 190 Cys Tyr Tyr Arg Met Glu Asn Ala Ser Leu Leu Leu SerSer Leu Ala 195 200 205 His Val Ser Lys Ala Val Gln Phe Leu Val Gln ThrThr Met Lys Arg 210 215 220 Leu Leu Ala His Arg Ser Leu Thr Glu Ile LeuLeu Glu Arg Lys Ser 225 230 235 240 Ile Ala Gln Asp Ala Lys Val Ala LeuAsp Ser Val Thr Cys Ile Trp 245 250 255 Gly Ile Lys Val Glu Arg Ile GluIle Lys Asp Val Arg Leu Pro Ala 260 265 270 Gly Leu Gln His Ser Leu AlaVal Glu Ala Glu Ala Gln Arg Gln Ala 275 280 285 Lys Val Arg Met Ile AlaAla Glu Ala Glu Lys Ala Ala Ser Glu Ser 290 295 300 Leu Arg Met Ala AlaGlu Ile Leu Ser Gly Thr Pro Ala Ala Val Gln 305 310 315 320 Leu Arg TyrLeu His Thr Leu Gln Ser Leu Ser Thr Glu Lys Pro Ser 325 330 335 Thr ValVal Leu Pro Leu Pro Phe Asp Leu Leu Asn Cys Leu Ser Ser 340 345 350 ProSer Asn Arg Thr Gln Gly Ser Leu Pro Phe Pro Ser Pro Ser Lys 355 360 365Pro Val Glu Pro Leu Asn Pro Lys Lys Lys Asp Ser Pro Met Leu 370 375 3803 1104 DNA Homo sapiens 3 aggatataaa ataagaaata cgtagggagg agagaaaggcatccttgaga cgactccaag 60 aaggaaagtt ggggatgagg cgaaatttct gattttaccttaaagtgacc ctaattcgat 120 gaccttttgt ggtttttttc ttttttcttt tttacttggccctgcccaag caggacctaa 180 aaacaaacag acaaaaaagg ttactaacaa ctgttcctctccacgaaaat ctgcagtaaa 240 aggtaaaaga tgtattcgtt ttgaagagaa accagagcttgcgatgagct tctgtatctc 300 cgtcagccct ctagcatgac attaggaacc ctccaggagatgagtcttca cagcccgggt 360 tggcacctgc agacacgcac ttttcaacgc ccgcaccctgcccggggccg gctctcccac 420 ccaggcctct ctctgcttca gcgccgcccc ggccgtgggagtcggcgggc gcagtccaca 480 gctccaccaa gacacagctg tcggggttcc gggtgcgccccgcccgcggc cccggtgtcc 540 cgcccctcgc cctcagcccc cacccgacgg tctttagggtcccccgggca cgccacgcgg 600 acccgcagcg actccacagg gactgcgctc ccgtgcccctagcgctcccg cgctgctgct 660 ccagccgccc ggcagctctg aggatggaga ggagggcgcggagctcctcc agggagtccc 720 gcgggcgagg cggcaggact ccgcacaagg agaacaagagggcaaaggcc gagaggagcg 780 gcgggggccg cgggcgccag gaggctgggc ccgagccgtcgggctccgga cgggcgggga 840 ccccggggga gccccgagcg cccgccgcca cggtggtggacgtggattag gtccgaggct 900 ccggcgagga gggcaccgag gtggtggcgc tgttggagagcgagcggccc gaggaaggta 960 cggattcagc accactatct gctacttttc caggtggtaactaaggggcg tcagataagg 1020 tggaaagggt catccccacg agacccactg aagccagagcagattgctgg atgctcaggt 1080 tcccaggaac ggaagggcgt aagt 1104 4 378 DNAHomo sapiens 4 tctgactact ctgatttgac ttattcctaa tatttccagc aaagtctccaagytgtgcca 60 actccaatac caagaattgg accaacagat gctagtcagt gaatacagtgaagtttcaat 120 ataattattg gtttgcttta atttttttaa ggtaccaaat cctccggcttaggggcctgt 180 gagtggcttc ttgtcctcat ttccctgctc ttcatcatca tgaccttccctttttccatc 240 tggttctgcg taaaggtgag attccataag gacccaatag gtaatttcctgaggcctctc 300 actggccaca ccatgcccat tctcacttct gttttctggt acatgttattgctccatgtg 360 gaatgccctc accccaga 378 5 700 DNA Homo sapiens 5catataaaag ctagtgcaga actcacagaa aatataagat ttaatatgcc ttgataagat 60taatttaggg aaagttggcc atggatttta gataatcata agtctttaat caaaattctg 120tctatgggtt caaaaattaa catggttaat atactttttc atttctgaaa ttttacactt 180actaaatata gattttggaa acttaagtat taatagaaat tttttcctgg ttctcaaaac 240aaaaaatttc tgatatctag gatcattctt atgccaaggc cttttgaaga ctttttcttt 300ctgggagtga tttgaaagga ttaaatttct ctttaggttg tacaagagta tgaaagagta 360attatattcc gactgggaca tctgcttcct ggaagagcca aaggccctgg taaaaaaaca 420ctcttttttt tctaaacacc tctctcctga cttgccaatt tcttcaaccc atgcagattt 480gtaatatgga cctcagatta aatgaagtaa cttgattcat gatatctgaa ttttccaatc 540tgttacttat aggttattca aatattcttc agagactatt actactaggt cataggtagc 600caagagagag aattggtaca gagagcccac atgccagggc aaggcttgct ggaatagcaa 660gttagcttag gaccaatggc tggggactga tgtgagtacg 700 6 360 DNA Homo sapiensmisc_feature 327 where n may be either a or g or c or t/u, unknown orother 6 ctggattaca tattataata tataatagtg ctctcctttt accctcaggtggaggtggga 60 tgggccaatg gtctgtaatt agaggctaag aaaagtaatg tagtgtgcaacctgacccca 120 gaaaggtgaa acccaaacag cyttcatgct agctatttat cygtcayttcctcctcctct 180 cttttaggtc ttttcttttt tttgccctgc ctggatacct accacaaggttgaccttcgt 240 ctccaaactc tggagatacc ttttcatgag gtaagccaaa tgatggcttttgctttctct 300 atacattttc catggtctac ctaccgngga caaaatgatt atttatactcaaaaatagga 360 7 649 DNA Homo sapiens 7 cttcttatga gaagattatt tctgattttttttacaaaag gatttaccac aggattaagt 60 tgtgcattct ttcgtgtatt taataaaaatttcataattt tcaaaaacat gtctatttta 120 aataaagggt aggccaactc catttttctcttgcggagaa aattcacttt gaacacattt 180 agttcctcta accccacata ggaaaggagcccaagaatca agcctgtcat ccaaactttt 240 ttctgcctag atcgtgacca aagacatgtttataatggag atagatgcca tttgctacta 300 ccgaatggaa aatgcctctc ttctcctaagcagtcttgct catgtatcta aagctgtgca 360 attccttgtg caaaccacta tgaagcgtctcctagcacat cgatccctca ctgaaattct 420 tctagagagg aagagcatcg cccaagatgcaaaggtactt agataaacat aatggccaat 480 atgctgaaat atttatcttt tattcatttgttcgttggac atttattaaa tcttctatgg 540 ccagttccat cccttagggg ccatccctttgggagctcat agctagttag gaggttgcca 600 aattgactct gagtcaatta tagttatcagtatggtgctt gttaatcag 649 8 592 DNA Homo sapiens 8 aattacattt agggccacctggataatcca ggataatatc cccatctcaa gagcctttaa 60 cctaatcaaa ccagcaaaatccctcttggg gtaacattca cagattccag ggattaggac 120 atgggatatc tttggggaccattattcagc ataccacacc atcttcaatt gcacagatat 180 ttattgggtg gcaccatgcaagttaaacaa ctctttgcaa ggcactgtga agttaaatac 240 aacaggcaaa taatgtcctttcaaagggaa tgttgttcct tagtacagaa caatggccac 300 cagggtttag gcatgctctcctcccacctg gaggctccca ctgacatctg aattcttctt 360 tccacaggtt gccttggattcagtgacctg tatttgggga atcaaagtgg agagaataga 420 aatgtgggta ggaaattaactagcaagaac tgtatgataa aggaaaatat tctggtttca 480 ttttaaattt ttcatttgaaaaattatttt cactgagtac tatagccata tcagcataaa 540 tttataaaaa agagaaacaaatcacctaat atcttacagc cataacacaa tc 592 9 716 DNA Homo sapiensmisc_feature 510 where n may be either a or g or c or t/u, unknown orother 9 cattgttcaa atttattagt tggggcttag attatatcct aagcggaaaaactgagcaca 60 gctcatcaaa tacaaaacct gctgtgctga taatgagaaa ctacagctctactgtagcat 120 cagcaataat acaaaactgc atttgaggca tcgaccttgg agatctgcctacttttgacc 180 tcagaagtct aggaatggca cactctggtc actccaaatt tgctactcatcatgagacag 240 cagtagagag gcttgcaagt ctgtgtgaaa gctttggccc ctaaatcatggctgcacacc 300 tacatacctg cattctttct ttttcagtaa agatgtgagg ttgccagctgggcttcagca 360 ctcactggct gtggaggctg aagcgcaaag acaagccaaa gtgcgggtgagcactccatc 420 ctcccaccca gactgccctt taggaaggcc tgctcgtgga gaacatttcccctttgcttc 480 cttactgtcc attcattagg cactgggcan aagctgtctt gggcccttacaactctatta 540 aaattgctct cttaaagtgt gttaatagtc ccctgactaa tgcaactcctctccctctct 600 gaagctactg ataatagtga ccactcactg cttgagtctc accttccctctctctcctta 660 aaggcatctc ctccacacac atcaatccct cttctctagt gctggcatcttttcct 716 10 1305 DNA Homo sapiens 10 ggcaaaatcc taatctttca aggcccaccagatgctaata actcccctaa tacttcattt 60 atacttgtga tggctcctaa cgcattccaccttaaattgt gattaacagt ttaatctgtc 120 tccccagctc aagacccttc agaaagaagaataaacatgt tctatgctta accgtgcttg 180 ccacatagta gatgctcagt gcttgtctgctgagtcatac tgcatagtgg tgaagccttc 240 agggaatgaa gaacaatcac tttgctttcgtcacatgttt tctagatgat tgctgcagaa 300 gcggaaaagg ctgcttctga gtccctgaggatggcagctg agattctgtc aggcacccct 360 gctgctgttc agcttcgata cctccacacccttcagtctc tgtccacaga gaagccttcc 420 actgtggttt tacctttgcc atttgacctactgaattgcc tgtcttctcc cagcaacaga 480 actcagggaa gcctcccctt cccaagtccttccaaacctg ttgagccact aaatcctaaa 540 aagaaagact ctcccatgtt ataggaaggatggggcataa tgtgactgta aaggggcctg 600 ccatagaaaa gtcacatccc tgagggagacactctgtcct cattccctgc ccttcctttg 660 gttgccatat ggaatggcca tggaatgcacgaagtcacaa tgcaccatcc atgagaagac 720 rgtgaaatga tgtaatgaca gagaaggcagacaacatgtt tccgtgactc atctagtcag 780 agcaattatg ggaaacagct ttggtcaacattctactttg graagaattt tgagtctaga 840 tgtggttaaa ttttgacttc tgggaacttggttcagatgt ccctttcact gtatgtcctc 900 tgaccccttt ggcaaggttg ccacagctcccacagccctt cctacaagca cctatcattg 960 ggcttgtcac actctattgc tcttctgtcccraagatgca gtcttctctc caatgatact 1020 accaagtctt agttttcctc aaccacactcaatctttttg ctccaccctg aattcctcac 1080 acctaaccct gatagttacc taaagtgacacttaaatgtt tcagagtgaa tgcaaaaaag 1140 agagatgtac ttggagtcgg atatacaatttatccctaat taaagcattt aaaaggaatt 1200 ctttttgtgg agattccttt tttaaacaaataaataaaag gacaaaaaca tctgacacat 1260 gtggcttaaa atctgaggga gaatcactataaatagtggg ccaga 1305 11 1065 DNA Homo sapiens 11 ccactatgaa gcgtctcctagcacatcgat ccctcactga aattcttcta gagaggaaga 60 gcatcgccca agatgcaaaggttgccttgg attcagtgac ctgtatttgg ggaatcaaag 120 tggagagaat agaaattaaagatgtgaggt tgccagctgg gcttcagcac tcactggctg 180 tggaggctga agcgcaaagacaagccaaag tgcggatgat tgctgcagaa gcggaaaagg 240 ctgcttctga gtccctgaggatggcagctg agattctgtc aggcacccct gctgctgttc 300 agcttcgata cctccacacccttcagtctc tgtccacaga gaagccttcc actgtggttt 360 tacctttgcc atttgacctactgaattgcc tgtcttctcc cagcaacaga actcagggaa 420 gcctcccctt cccaagtccttccaaacctg ttgagccact aaatcctaaa aagaaagact 480 ctcccatgtt ataggaaggatggggcataa tgtgactgta aaggggcctg ccatagaaaa 540 gtcacatccc tgagggagacactctgtcct cattccctgc ccttcctttg gttgccatat 600 ggaatggcca tggaatgcacgaagtcacaa tgcaccatcc atgagaagac agtgaaatga 660 tgtaatgaca gagaaggcagacaacatgtt tccgtgactc atctagtcag agcaattatg 720 ggaaacagct ttggtcaacattctactttg gaaagaattt tgagtctaga tgtggttaaa 780 ttttgacttc tgggaacttggttcagatgt ccctttcact gtatgtcctc tgaccccttt 840 ggcaaggttg ccacagctcccacagccctt cctacaagca cctatcattg ggcttgtcac 900 actctattgc tcttctgtcccgaagatgca gtcttctctc caatgatact accaagtctt 960 agttttcctc aaccacactcaatctttttg ctccaccctg aattcctcac acctaaccct 1020 gatagttacc taaagtgacacttaaatgtt tcagagtgaa tgcaa 1065 12 20 DNA Homo sapiens 12 gcagcgactccacagggact 20 13 20 DNA Homo sapiens 13 tcagtgggtc tcgtggggat 20 14 20DNA Homo sapiens 14 aggcagtgaa tacagtgaag 20 15 19 DNA Homo sapiens 15ggcctcagga aattaccta 19 16 20 DNA Homo sapiens 16 ttctgggagt gatttgaaag20 17 20 DNA Homo sapiens 17 tgaagaaatt ggcaagtcag 20 18 19 DNA Homosapiens 18 aaggtgaaac ccaaacagc 19 19 20 DNA Homo sapiens 19 cggtaggtagaccatggaaa 20 20 20 DNA Homo sapiens 20 cataggaaag gagcccaaga 20 21 20DNA Homo sapiens 21 tttcagcata ttggccatta 20 22 17 DNA Homo sapiens 22ctcccactga catctga 17 23 20 DNA Homo sapiens 23 aatttaaaat gaaaccagaa 2024 20 DNA Homo sapiens 24 ctaaatcatg gctgcacacc 20 25 20 DNA Homosapiens 25 cttcctaaag ggcagtctgg 20 26 20 DNA Homo sapiens 26 ggtgaagccttcagggaatg 20 27 20 DNA Homo sapiens 27 ttctatggca ggccccttta 20 28 1652DNA Rattus rattus CDS (47)..(1195) 28 cgactctgcc agcatctggc tttggggggcgtgcccgccg cgtaga atg gac agc 55 Met Asp Ser 1 agg gcg cgg agc tct tccaga aag acc cac ggg aga ggt agc agg tcc 103 Arg Ala Arg Ser Ser Ser ArgLys Thr His Gly Arg Gly Ser Arg Ser 5 10 15 tct tct agg gat gac aag aagtca aag gcc ggg agg ggc aac aga ggc 151 Ser Ser Arg Asp Asp Lys Lys SerLys Ala Gly Arg Gly Asn Arg Gly 20 25 30 35 cgc gcg cgc ccg gat gct ggggca gag cgg cag agc gcc ggg cgg acg 199 Arg Ala Arg Pro Asp Ala Gly AlaGlu Arg Gln Ser Ala Gly Arg Thr 40 45 50 ggg acc cgg gag gag cac cga gctcca gca gcc acg gta gtg aat gtg 247 Gly Thr Arg Glu Glu His Arg Ala ProAla Ala Thr Val Val Asn Val 55 60 65 gac gag gtt cga agc ccg ggt gag gagggt acg gaa gtg gtg gcc ctg 295 Asp Glu Val Arg Ser Pro Gly Glu Glu GlyThr Glu Val Val Ala Leu 70 75 80 ctg gag agc gag cga cca gag gaa ggg atcaag ccc tct gga tta ggg 343 Leu Glu Ser Glu Arg Pro Glu Glu Gly Ile LysPro Ser Gly Leu Gly 85 90 95 gcc tgc gag tgg ctt ctt gtc ctc tcc tcc ctgatc ttc atc atc gta 391 Ala Cys Glu Trp Leu Leu Val Leu Ser Ser Leu IlePhe Ile Ile Val 100 105 110 115 acg ttt ccc ttt tcc atc tgg ttc tgc ataaag gtt gtt caa gaa tac 439 Thr Phe Pro Phe Ser Ile Trp Phe Cys Ile LysVal Val Gln Glu Tyr 120 125 130 gaa aga gta att ata ttc cga ctg gga catctg ctt cct gga aga gcc 487 Glu Arg Val Ile Ile Phe Arg Leu Gly His LeuLeu Pro Gly Arg Ala 135 140 145 aaa gga cct ggc ctg ttc ttt ttt cta ccctgc ctg gac acc tat cac 535 Lys Gly Pro Gly Leu Phe Phe Phe Leu Pro CysLeu Asp Thr Tyr His 150 155 160 aag gtt gac ctc cgt ctc cag acc ttg gaaata cct ttc cat gag gtg 583 Lys Val Asp Leu Arg Leu Gln Thr Leu Glu IlePro Phe His Glu Val 165 170 175 gta acc aaa gat atg ttc aca atg gag atagac gct gtc tgc tac tac 631 Val Thr Lys Asp Met Phe Thr Met Glu Ile AspAla Val Cys Tyr Tyr 180 185 190 195 cgc atg gaa aat gcc tcc ctt ctt ctaagc agt cta gct cat gtg tcc 679 Arg Met Glu Asn Ala Ser Leu Leu Leu SerSer Leu Ala His Val Ser 200 205 210 aaa gcc atc cag ttc ctg gtg caa accacc atg aag cgc ctc ttg gca 727 Lys Ala Ile Gln Phe Leu Val Gln Thr ThrMet Lys Arg Leu Leu Ala 215 220 225 cat cga tcc ctc act gaa att ctc ctggaa agg aag agc att gcc caa 775 His Arg Ser Leu Thr Glu Ile Leu Leu GluArg Lys Ser Ile Ala Gln 230 235 240 gat gta aag gtt gcc ttg gac tca gtgacc tgt gtt tgg ggc atc aaa 823 Asp Val Lys Val Ala Leu Asp Ser Val ThrCys Val Trp Gly Ile Lys 245 250 255 gtg gag aga act gaa att aag gat gtacgg ctg cca gct ggg ctt cag 871 Val Glu Arg Thr Glu Ile Lys Asp Val ArgLeu Pro Ala Gly Leu Gln 260 265 270 275 cac tct ctg gct gtg gaa gct gaggca caa aga cag gcc aaa gtg cgg 919 His Ser Leu Ala Val Glu Ala Glu AlaGln Arg Gln Ala Lys Val Arg 280 285 290 gtg att gct gcc gaa ggg gaa aaagct gcc tct gag tcc ctg agg atg 967 Val Ile Ala Ala Glu Gly Glu Lys AlaAla Ser Glu Ser Leu Arg Met 295 300 305 gcg gct gag att ctg tca ggc acccca gct gct gtc cag ctc cgg tac 1015 Ala Ala Glu Ile Leu Ser Gly Thr ProAla Ala Val Gln Leu Arg Tyr 310 315 320 ctg cac act ctt cag tcc ttg tccaca gac aag ccg tcc acc gtg gtt 1063 Leu His Thr Leu Gln Ser Leu Ser ThrAsp Lys Pro Ser Thr Val Val 325 330 335 ttg cct tta ccc ttt gac atg ctgaac ctt ctc tcc tct ccc agc aac 1111 Leu Pro Leu Pro Phe Asp Met Leu AsnLeu Leu Ser Ser Pro Ser Asn 340 345 350 355 aga gca caa gga agc atc aactac cca agt tct ccc aaa cct gtt gaa 1159 Arg Ala Gln Gly Ser Ile Asn TyrPro Ser Ser Pro Lys Pro Val Glu 360 365 370 cca cta aat ccc aaa agg aaggac tct cct atg cta taggggcgag 1205 Pro Leu Asn Pro Lys Arg Lys Asp SerPro Met Leu 375 380 tggacaagag taatgggaat acaccatata aagccgtatccctgagcgag gcattcggtc 1265 cccacgccca ggcccaccct gcccttgttg tttgccttttgagtgtatca tgtcacaaga 1325 tggacacacg catgagaaca cagtgaaatg gcagagaagacatccagcca cacaagtggg 1385 tcgtctcatc attcattaca ggaaagaaag agatttagaattttgggttg aggggctgga 1445 gagatggctc agtggttatg aacactgact gctcttccagaggtcctgag ttcaaatccc 1505 agcaaccaca tggtggctca caaccatctg taatgggatccgatgccctc ttctggtgtg 1565 taagacagtg acagtgtact catcatatat aagatgaataaataaaccct tttaaaaaaa 1625 aaaaaaaaaa aaaaaaaaaa aaaaaaa 1652 29 383 PRTRattus rattus 29 Met Asp Ser Arg Ala Arg Ser Ser Ser Arg Lys Thr His GlyArg Gly 1 5 10 15 Ser Arg Ser Ser Ser Arg Asp Asp Lys Lys Ser Lys AlaGly Arg Gly 20 25 30 Asn Arg Gly Arg Ala Arg Pro Asp Ala Gly Ala Glu ArgGln Ser Ala 35 40 45 Gly Arg Thr Gly Thr Arg Glu Glu His Arg Ala Pro AlaAla Thr Val 50 55 60 Val Asn Val Asp Glu Val Arg Ser Pro Gly Glu Glu GlyThr Glu Val 65 70 75 80 Val Ala Leu Leu Glu Ser Glu Arg Pro Glu Glu GlyIle Lys Pro Ser 85 90 95 Gly Leu Gly Ala Cys Glu Trp Leu Leu Val Leu SerSer Leu Ile Phe 100 105 110 Ile Ile Val Thr Phe Pro Phe Ser Ile Trp PheCys Ile Lys Val Val 115 120 125 Gln Glu Tyr Glu Arg Val Ile Ile Phe ArgLeu Gly His Leu Leu Pro 130 135 140 Gly Arg Ala Lys Gly Pro Gly Leu PhePhe Phe Leu Pro Cys Leu Asp 145 150 155 160 Thr Tyr His Lys Val Asp LeuArg Leu Gln Thr Leu Glu Ile Pro Phe 165 170 175 His Glu Val Val Thr LysAsp Met Phe Thr Met Glu Ile Asp Ala Val 180 185 190 Cys Tyr Tyr Arg MetGlu Asn Ala Ser Leu Leu Leu Ser Ser Leu Ala 195 200 205 His Val Ser LysAla Ile Gln Phe Leu Val Gln Thr Thr Met Lys Arg 210 215 220 Leu Leu AlaHis Arg Ser Leu Thr Glu Ile Leu Leu Glu Arg Lys Ser 225 230 235 240 IleAla Gln Asp Val Lys Val Ala Leu Asp Ser Val Thr Cys Val Trp 245 250 255Gly Ile Lys Val Glu Arg Thr Glu Ile Lys Asp Val Arg Leu Pro Ala 260 265270 Gly Leu Gln His Ser Leu Ala Val Glu Ala Glu Ala Gln Arg Gln Ala 275280 285 Lys Val Arg Val Ile Ala Ala Glu Gly Glu Lys Ala Ala Ser Glu Ser290 295 300 Leu Arg Met Ala Ala Glu Ile Leu Ser Gly Thr Pro Ala Ala ValGln 305 310 315 320 Leu Arg Tyr Leu His Thr Leu Gln Ser Leu Ser Thr AspLys Pro Ser 325 330 335 Thr Val Val Leu Pro Leu Pro Phe Asp Met Leu AsnLeu Leu Ser Ser 340 345 350 Pro Ser Asn Arg Ala Gln Gly Ser Ile Asn TyrPro Ser Ser Pro Lys 355 360 365 Pro Val Glu Pro Leu Asn Pro Lys Arg LysAsp Ser Pro Met Leu 370 375 380

1. An isolated nucleic acid, the sequence of which is chosen from SEQ IDno 3 to SEQ ID no 10, or a homologous sequence defined as i) a sequencewhich is identical to at least 70% of the sequence SEQ ID no 3 to SEQ IDno 10; or ii) a sequence which hybridizes with the sequence SEQ ID no 3to SEQ ID no 10, or the sequences complementary thereto, under stringenthybridization conditions.
 2. An isolated polypeptide, named podocin,comprising the amino acid sequence SEQ ID no 2 or no 29, or a homologoussequence defined as i) a sequence which is identical to at least 70% ofthe sequence SEQ ID no 2 or no 29; or ii) a sequence which is encoded bya nucleic acid sequence which hybridizes with the sequence SEQ ID no 1or no 28, or the sequences complementary thereto, under stringenthybridization conditions.
 3. An isolated nucleic acid comprising asequence which differs from the sequence SEQ ID no 1 by mutation,insertion or deletion, in particular in at least one of the positions ofnucleotides 481, 173/174, 488, 924/925, 128, 343, 482, 548, 607 and 940,or else 1033, 529, 622, 774-782, 154, 422, 442, 571, 572, 583, 783, 794and
 848. 4. An isolated nucleic acid comprising the sequence SEQ ID no 1or no 28, or a homologous sequence defined as i) a sequence which isidentical to at least 70% of the sequence SEQ ID no 1 or no 28; or ii) asequence which encodes the polypeptide, named podocin, as defined inclaim
 2. 5. A cloning and/or expression vector containing a nucleic acidas claimed in one of claims 1, 3 and
 4. 6. A host cell transfected witha vector as claimed in claim
 5. 7. A nucleic acid consisting of asequence chosen from the sequences SEQ ID no 11 to SEQ ID no
 27. 8. Amethod for producing a recombinant podocin polypeptide, in which avector containing a nucleic acid as claimed in claim 4 is transferredinto a host cell, which is cultured under conditions which allowexpression of the polypeptide as claimed in claim
 2. 9. An antibodydirected against the polypeptide as defined in claim
 2. 10. The use ofat least one antibody as claimed in claim 9, for detecting or purifyinga polypeptide as defined in claim 4 in a biological sample.
 11. The useof at least one nucleic acid as claimed in claim 1, 3, 4 or 7, fordetecting an abnormality in the NPHS2 gene, defined as comprising anucleic acid sequence as claimed in claim 1, or in its transcript,defined as comprising a nucleic acid sequence complementary to thesequence of claim
 4. 12. A method for the in vitro diagnosis of asteroid-resistant nephrotic syndrome, comprising the steps consistingin: a1) placing a biological sample containing DNA together withspecific oligonucleotides for amplifying all or part of the NPHS2 gene,defined as comprising a nucleic acid sequence as claimed in claim 1; b1)amplifying said DNA; c1) detecting the amplification products; d1)comparing the amplification products obtained with those obtained usinga control sample, and detecting in this way a possible abnormality insaid NPHS2 gene, indicating a steroid-resistant nephrotic syndrome; or,according to an alternative, a2) placing a biological sample containingRNA together with specific oligonucleotides for amplifying all or partof the transcript of the NPHS2 gene, defined as comprising a nucleicacid sequence complementary to the sequence of claim 4; b2) amplifyingsaid DNA; c2) detecting the amplification products; d2) comparing theamplification products obtained with those obtained using a controlsample, and detecting in this way a possible abnormality in saidtranscript of the NPHS2 gene, indicating a steroid-resistant nephroticsyndrome.
 13. A pharmaceutical composition comprising a polypeptide asclaimed in claim 2 or a nucleic acid encoding said polypeptide, incombination with a pharmaceutically acceptable vehicle.
 14. The use of apolypeptide as claimed in claim 2 or of a nucleic acid encoding saidpolypeptide, for producing a medicinal product intended for thetreatment of renal diseases, in particular a steroid-resistant nephroticsyndrome.